The incidence of sexually transmitted infections (STI) continues to be a global health problem. New and novel strategies are needed to reduce the incidence of STI, particularly in women as they often suffer from severe medical complications as a result of such infections. Our long-term goal is to understand the mechanisms of the innate host defense system that functions at mucosal surfaces in response to Neisseria gonorrhoeae. Antimicrobial peptides (AP) produced by phagocytic and epithelial cells are now recognized as important mediators of innate immunity. Due to their potent activity in vitro, we hypothesize that they could be engineered as topical microbicides for use in the prevention of STI. Towards that end, we are interested in how these peptides exert antigonococcal activity in vitro and how gonococci might subvert their antibiotic-like action through genetic and physiologic processes. The human AP termed LL-37 has been shown by us to exert potent action in vitro against N. gonorrhoeae and can block infection by Treponema pallidum in a rabbit skin lesion model. We have recently engineered truncated versions of LL-37 that maintain antigonococcal activity in vitro without killing lactobacilli, which are important members of the vaginal normal flora. In order to understand how LL-37 contributes to innate immunity during STI, we will design nontoxic variants of LL-37 that have enhanced capacities to kill bacterial pathogens (N. gonorrhoeae, Haemophilus ducreyi, T. pallidum and Chlamydia trachomatis) in vitro and in vivo but remain inactive against lactobacilli (Specific Aim 1). As is the case with other antibiotics, it is likely that bacterial pathogens can develop mechanisms to resist the action of AP. We will use the gonococcus as a model STI pathogen to examine this issue in order to identify the genes that may endow bacteria with the ability to constitutively (Specific Aim 2) or inducibly (Specific Aim 3) resist the action of LL-37.We will use transposon mutagenesis techniques in Specific Aim 2 and the micro array technology in Specific Aim 3 to identify these determinants. The results from our studies should generate novel compounds that could be used as topical microbicides to block STI, provide a better understanding of innate immunity at the genital mucosal surface during STI and result in important insights regarding bacterial responses to AP in vivo.